Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes.

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call "flicker." These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.

Mechanisms of K+ uptake across the basal membrane of malpighian tubules of Formica polyctena: the effect of ions and inhibitors.

In the presence of 6 mmol l-1 Ba2+, known to block the K+ channels in the basal membrane, a rise in bath [K+] ([K+]bl) induced an increase in intracellular K+ concentration ([K+]i) similar in amount and in time course to that obtained in the absence of Ba2+. The presence of active and passive (other than through K+ channels) K+ uptake mechanisms across the basal membrane was investigated in different bath K+ concentrations. Dihydro-ouabain (10(-3) mol l-1), a blocker of the Na+/K(+)-ATPase, tested in low bath [K+], and Sch28080 (10(-4) mol l-1), a K+/H(+)-ATPase inhibitor, were without effect on fluid secretion. Dihydro-ouabain was also without effect on electrical potential differences either in the absence or in the presence of Ba2+. Vanadate (10(-3) mol l-1), in contrast, strongly reduced fluid secretion not only in control solution but also in high-K+, Na(+)-free medium and reduced the transepithelial and the apical membrane potential differences but not the basal membrane potential difference of [K+]i. Omitting Na+ from the bathing medium, replacing Cl- by Br- or applying bumetanide (10(-5) mol l-1) inhibited fluid secretion only in a low-K+ (10 mmol l-1) medium. In 51 mmol l-1 [K+]bl, omitting Na+ was without effect and 10(-4) mol l-1 bumetanide was needed to inhibit secretion. Replacing Cl- by Br- stimulated fluid secretion at this K+ concentration. Bumetanide (10(-4) mol l-1) had no effect in 113 mmol l-1 [K+]bl. Bumetanide (10(-4) mol l-1) in 51 mmol l-1 [K+]bl did not affect membrane potentials, did not lower [K+]i and did not affect the rise in [K+]i observed on an increase in [K+]bl. The results were summarized in a model proposing that K+ channels play a dominant role in high-K+ (113 mmol l-1) bathing medium. A K+/Cl- cotransporter may become more important in 51 mmol l-1 [K+]bl and a K+/Na+/2Cl- cotransporter may gain in importance in 10 mmol l-1 [K+]bl. Active mechanisms for K+ uptake across the basal membrane seem to play no detectable role in sustaining fluid secretion. The response to vanadate might be due to an effect on the apical electrogenic H+ pump.

Electrophysiological evidence for the presence of an apical H(+)-ATPase in Malpighian tubules of Formica polyctena: intracellular and luminal pH measurements.

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K+ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H+ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K+) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K+ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K+ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from -93 mV in 5 mmol/l K+ to -65 mV in 113 mmol/l K+. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K+ concentration. The calculated electromotive force of this pump is -159 mV. Comparing the proton with the K+ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K+ extrusion into the lumen for each value of secretion rate.

Block of the transient inward current by R56865 in guinea-pig ventricular myocytes.

Single myocytes isolated from the guinea-pig ventricle were used under voltage-clamp conditions to study the activity of a newly developed drug, R56865, a benzothiazolamine derivative, against digitalis toxicity. Multicellular Purkinje preparations were used in some experiments. The drug blocked the transient inward current (TI) with an apparent KD of 7.5 X 10(-8) M. The blockade was not due to a direct effect on the Na+, Ca2+ exchange current or on the non-selective cation current, the two components of TI. Blockade of TI was accompanied in time with inhibition of after-contractions, which suggests that the drug has an effect on Ca(2+)i transients. At concentrations effective on TI the drug did not block Ca2+ currents or Na+ currents; however the Na+ current was inhibited after the cells had been pretreated with DPI 201-106. It is suggested that the inhibitory effect on TI may be due to an effect on the release of Ca2+ from the sarcoplasmic reticulum or to a reduction in Ca2+ overload via blockade of the Na+ channel.